Breeding for compositionally enhanced agricultural products can require the analysis of a large number of seed samples from plants to identify those plants with the desired compositional and agronomic properties for use or advancement to the next generation. Analysis of bulk seed batches for certain traits, such as high oil or protein content, on a single plant or ear, in conjunction with an appropriate breeding methodology such as recurrent selection, often allows for the selection and introduction of such traits into a commercial population. Although the analysis of these seed batches can be performed by various techniques, methods that are rapid, low cost, and non-destructive are the most desirable.
Magnetic resonance imaging (MRI) is based on a non-invasive spectroscopic technique known as nuclear magnetic resonance (NMR). NMR requires the sample under investigation to contain atoms that exhibit nuclear spin, an intrinsic quality makes the atomic nuclei magnetic. The most common atom with nuclear spin is hydrogen, whose nucleus is a proton with spin ½. A typical proton NMR (1H NMR) experiment involves placing a sample to be studied in a strong homogeneous magnetic field. The strong magnetic field causes preferential alignment of the protons in the sample with the magnetic field, a phenomenon that is analogous to a magnetic compass needle aligning with the earth's magnetic field. In a simple modern “pulsed-NMR” experiment, another magnetic field (the radiofrequency, or RF field) is transiently applied to the sample, which has the effect of rotating the aligned protons to a higher energy state, which is perpendicular to the strong magnetic field (this is called a 90° pulse). The protons precess at characteristic rates as they realign with the strong field, thus inducing a current in a pair of coils that serve as the detector. The current is measured in the detection coils as a function of time, and from this the rates of precession of the protons in the sample are inferred. The rates of precession are determined primarily by the strength of the strong magnetic field (with stronger fields leading to greater precession rates), but the unique molecular environment experienced by each of the protons also has an effect on the rate of precession. It is the unique molecular environment that is the object of study in NMR experiments.
In a slightly more complicated modern pulsed NMR experiment, the complexity of the signals arising from multiple molecular environments can be eliminated, leaving only the signals from protons in a selected subset of molecular environments. This approach, called the “spin-echo” experiment, can be used, for example, on a sample comprising a mixture of oil and water to eliminate the signal arising from the protons in water molecules, and to leave only signals arising from protons in oil molecules. This can be accomplished by using a series of RF pulses of 90° and 180°, and commercial instruments typically come with software that is useful for programming the instrument to properly execute the pulse sequence.
Non-imaging NMR-based technology for measuring characteristics such as oil content in intact seeds requires measurement of bulk samples and is unable to distinguish characteristics of individual seeds. These low-field pulsed NMR methods rely on differentiation of oil from other components in the seed based on inherent differences in longitudinal and spin-spin nuclear relaxation rates between oil and other protonated species. NMR methods used to measure oil have been standardized, they are non-destructive, robust, and they yield both accurate and reproducible results. But low-field NMR methods are performed at low magnetic field strengths, which yields poor signal-to-noise relative to the MRI experiment, and consequently, these methods offer a poor approach for single-seed analysis. The MRI method for measuring oil content is based on the same physical principles described in pulsed NMR studies. The difference is that MRI provides spatially encoded NMR signals, providing displays of data in an image format rather than a conventional NMR spectrum.
Another similarity exists between conventional NMR studies and the MRI method for measuring oil levels in single seeds. Both methods provide a relative oil content for seeds on a percent basis (wt/wt) by comparing the experimental results with oil calibration standards, which is the generally accepted and useful method for comparing oil levels in single seeds. See Tiwari et al., “Rapid and nondestructive determination of seed oil by pulsed nuclear magnetic resonance technique,” J. Am. Oil Chem. Soc. 51: 104-109 (1974). Relative numbers are obtained because a small portion of the NMR signal intensity is lost during the timing delays incorporated into the data acquisition schemes. The relative numbers can be corrected to absolute numbers by normalizing the data using an independent oil measurement for a given population of seeds, e.g., the average of the measured oil value for the population is set to the oil value determined in a bulk measurement and all individual seeds are adjusted accordingly. Absolute oil numbers also can be obtained using NMR spectroscopy and MRI methods by using seed standards in the analysis. In this case, the oil content of a seed is determined absolutely since the seed standard more closely matches the nuclear relaxation and wax content of the experimental seed. However, absolute oil numbers have been shown to be less precise for comparing relative oil levels between seeds. See Rubel, “Simultaneous determination of oil and water contents in different oilseeds by pulsed nuclear magnetic resonance,” J. Am. Oil Chem. Soc. 71: 1057-1062 (1994).
While the best NMR instruments require extremely homogenous magnetic fields, MRI machines purposely induce field gradients (variations in magnetic field strength with respect to position) using three gradient magnets. The gradient magnets are much weaker than the strong. magnet, but they are sufficient to cause distinguishable proton precession rates at different parts of the sample. Modern MRI machines pulse not only the RF field, but also the gradient fields, in order to selectively rotate and selectively detect protons in particular regions of the sample. Measurement of the signal strength of the various frequency components (precession rates) indicates the density and relaxation times (the time it takes particular protons to relax back to their original low-energy state and re-align with the strong magnetic field, the longitudinal relaxation time, and to precess out of phase, or dephase due to spin-spin relaxation, with respect to the other protons) of protons at various locations in the sample.
MRI methods provide direct measurements of characteristics such as oil levels, thus providing a primary assay independent of a chemometric model. Thus a three dimensional image can be constructed where the intensities at various points in the image relate to the densities and relaxation times of protons at those points in the sample. Furthermore, because different molecular environments result in different precession frequencies, the molecular environment at each point in the sample can be determined. Such an approach is called “chemical shift imaging” (CSI).
An alternative to CSI is “spin echo imaging” (SEI). In this experiment, one class of protons can be singled out (based on their dephasing rates), and the signals from all other protons can be suppressed. For example, if a sample contains spatially separated oil and water, and a SEI experiment is directed to detecting the oil, then the resulting image will show only those regions of the sample in which oil is found.
MRI is a well-known non-invasive radiological technique commonly used in the medical sciences. The long-wavelength (radio wave) radiation is universally regarded as less harmful than the forms of radiation used in other types of non-invasive radiological techniques such as X-ray CAT (computed axial tomography) scans. As early as 1988, MRI techniques were starting to be applied to the study of plants. See Introduction section of Lakshminarayana et al., “Spatial distribution of oil in groundnut and sunflower seeds by nuclear magnetic resonance imaging,” J. Biosci. 17(1): 87-93 (March 1992) (hereinafter Lakshminarayana et al.) (describing a history up to 1992 of the use of MRI in the study of plants, seeds, and plant tissue). Lakshminarayana et al. describe an experiment in which MRI was used on single seeds to determine the spatial distribution of oil and water in single seed samples. They used a spin echo pulse sequence to selectively detect only the protons that were part of oil molecules. MRI was also used to study water uptake in dry kidney beans by Heil et al., “Magnetic resonance imaging and modeling of water up-take into dry beans,” Lebensm.-Wiss. u.-Technol. 25:280 (1992).
Both SEI and CSI MRI were used to image lipids in pecan embryos by Halloin et al., “Proton magnetic resonance imaging of lipid in pecan embryos,” J. Am. Oil Chemists' Soc. 70:1259 (1993). These experiments studied the differences in the images of pecan embryos that were normal, infected by fungus, and damaged by insects. The CSI MRI experiment showed the distribution of lipids and water within the pecan embryos.
Other MRI techniques are known to those skilled in the art. One example is relaxography, or relaxation time mapping. In this technique, different regions of a sample being imaged are distinguished based on the differences in relaxation times of the protons in the different regions.
MRI experiments on seeds have traditionally been conducted using research grade MRI instruments, for example, the Bruker AMX-400 9.4 Tesla instrument with an 8.9 cm diameter bore (Bruker Instruments Inc., Billerica, Mass.) or the GE Omega system 7.1 Tesla instrument with a 15 cm diameter bore (General Electric, Milwaukee, Wis.). These instruments typically have a bore size of a few centimeters, and consequently can only be used to study samples that are smaller than the bore size. The advantage of the small scale of these instruments is that the detection coils are close to the sample under investigation, and therefore their sensitivity is very high. Larger research grade MRI instruments also exist with bore sizes and detection coil diameters of 20-50 cm, for example, the Bruker Biospec II 4.7 Tesla, 40 cm diameter bore (Bruker Instruments, Inc., Billerica, Mass.). These instruments are useful for imaging of mice and other small animals. Clinical MRI instruments, on the other hand, must have a bore size and detection coils that are large enough to accommodate a human body. For example, Siemens (Siemens AG, Erlangen, Germany) and GE Medical Systems (Milwaukee, Wis.) manufacture a wide range of clinical instruments that can accommodate objects with 50 cm diameters and larger. Some newer systems with permanent magnets with magnetic fields as low as 0.2 Tesla are not limited by bore size, but by coil diameter. But the larger diameter detection coils in traditional superconducting instruments, as well as newer permanent magnet systems, make these clinical instruments ill-suited for imaging small samples such as seeds. The large distance of the detection coils from a small sample and the inherently weak signal emanating from a small sample conspire to make conventional approaches to quantitative imaging of small samples using clinical MRI instruments impractical. But the small volumes of small bore size research MRI instruments do not allow the simultaneous imaging of as many seeds as a clinical MRI instrument would allow if the larger MRI instruments were amenable to detecting signals from small samples.
Conventional non-invasive methods for bulk seed analysis, such as infrared (IR) spectroscopy, suffer from the disadvantage of being rather time-intensive and producing results that may not be fairly representative of the sample analyzed. Time-intensive techniques for finding desirable characteristics are especially disadvantageous to selective plant breeding programs, where many single seeds need to be screened rapidly in order to allow seed selection before the next planting generation. Delays in providing the breeder with the analytical results can cause the loss of an entire breeding cycle.
Non-imaging techniques such as IR spectroscopy suffer from the further disadvantage of collecting information from only a subset of a total sample by spot sampling only portions of only a few seeds out of the hundreds of seeds in the bulk sample. Furthermore, since spot sampling interrogates arbitrary portions of the seed, different tissues of the seeds in the samples can be misrepresented by the analytical data. Since qualities like oil content are often present in different amounts in different tissues, non-imaging techniques can fail to accurately assess the desired quality. Non-imaging techniques disregard spatial information, and thus provide no information to a plant breeder about the size, shape, mechanical damage, insect infestation, or fungal damage.
Conventional seed analysis techniques also fail to provide an efficient method for single seed analysis, which can greatly accelerate the rate of varietal development. Single seed analysis is necessary to differentiate and select individual seeds from the heterogeneous population of seeds often encountered in breeding populations. Single seed analysis can reduce the number of generations required for the production of a plant with the desired trait. Single seed selection also reduces the number of individual plants required. In corn, for example, the ability to identify the individual seeds with the desired trait at the single seed level rather than at the whole ear level can reduce the nursery requirement by 100 fold. This makes it possible to conduct a far greater number of breeding projects with the same resources.
Other conventional analytical techniques, such as gas chromatography, also often fail to provide an efficient method for single seed analysis. For example, the conventional method for single seed analysis of canola requires manual excision of one half of each seed for fatty acid analysis by gas chromatography, while the other half is planted. Because of the manual sample preparation and the low throughput of this analytical technique, only a small number of samples can be run per hour using this process. Furthermore, this technique allows for the possibility of destroying analyzed seeds' potentials to grow into mature, seed bearing plants.
Although single seed analysis is desirable, conventional approaches and sampling methods do not allow for efficient processing of single seeds. Conventional techniques require extensive manual input, which limits the rate of development of plants with improved characteristics.
Conventional spectroscopic analysis techniques do not allow for the localization of chemical component levels within different tissues of seeds. Conventional approaches, such as manual dissection of the seed followed by chemical analysis by traditional analytical techniques, are not only laborious and destructive, they also result in poor resolution of the components and poor quantitation, since the sample size resulting from dissection of individual seeds is below the sample size at which most traditional techniques produce reliable results.
Needed in the art are devices and methods for rapid analysis of bulk and single seeds that can efficiently and non-destructively analyze the morphological and/or chemical characteristics of individual seeds, and that can be integrated into an agricultural processing machine. The present invention provides such devices and methods.